Article

Structural determination of wild-type lactose permease. Proc Natl Acad Sci USA

Department of Physiology, University of California, Los Angeles, CA 90095-1662, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2007; 104(39):15294-8. DOI: 10.1073/pnas.0707688104
Source: PubMed

ABSTRACT

Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H(+) binding. To initiate transport, binding of sugar and/or an H(+) electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.

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Available from: Osman Mirza
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    • "The lactose permease of Escherichia coli (LacY) with 12 mostly irregular transmembrane a helices organized into two pseudosymmetrical six-helix bundles connected by a relatively long cytoplasmic loop (Abramson et al., 2003; Chaptal et al., 2011; Guan et al., 2007; Kumar et al., 2014; Mirza et al., 2006) is a paradigm for the major facilitator superfamily (MFS) (Marger and Saier, 1993). In the native membrane, which is 70%–80% zwitterionic phosphatidylethanolamine (PE) and 20%–25% anionic phosphatidylglycerol (PG) plus cardiolipin, LacY adopts a native topology with the N and C termini on the cytoplasmic surface of the membrane (Bogdanov et al., 2008; Calamia and Manoil, 1990; Chen and Wilson, 1984; Foster et al., 1983; Seto-Young et al., 1985). "
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    ABSTRACT: Lipids of the Escherichia coli membrane are mainly composed of 70%-80% phosphatidylethanolamine (PE) and 20%-25% phosphatidylglycerol (PG). Biochemical studies indicate that the depletion of PE causes inversion of the N-terminal helix bundle of the lactose permease (LacY), and helix VII becomes extramembranous. Here we study this phenomenon using single-molecule force spectroscopy, which is sensitive to the structure of membrane proteins. In PE and PG at a ratio of 3:1, ∼95% of the LacY molecules adopt a native structure. However, when PE is omitted and the membrane contains PG only, LacY almost equally populates a native and a perturbed conformation. The most drastic changes occur at helices VI and VII and the intervening loop. Since helix VII contains Asp237 and Asp240, zwitterionic PE may suppress electrostatic repulsion between LacY and PG in the PE:PG environment. Thus, PE promotes a native fold and prevents LacY from populating a functionally defective, nonnative conformation. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Mar 2015 · Structure
    • "Solving the structure of LacY was of particular impor - tance , as LacY represents the best - characterized MFS family member thus far ( Kumar et al . , 2014 ; Madej et al . , 2014 ) . To date , seven different crystal structures of wild - type and mutant LacY have been determined ( Abramson et al . , 2003 ; Mirza et al . , 2006 ; Guan et al . , 2007 ; Chaptal et al . , 2011 ; Kumar et al . , 2014 ) . The LacY structures revealed that the 12 TM helices are indeed arranged in two domains of six TM helices ( N - and C - domains ) , which have an identical topology and are related by a pseudo - twofold symmetry axis running nearly perpendicular to the mem - brane . As highlighted in Fi"
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    ABSTRACT: Abstract The recently increasing number of atomic structures for active transporters has not only revealed a strong conservation in the architecture of sequence-unrelated transporter families, but also identified a unifying element called the "inverted repeat topology", which is found nearly in all transporter folds to date. Indeed, most membrane transporters consist of two or more domains with similar structure, so-called repeats. It is tempting to speculate that transporters have evolved by duplication of one repeat followed by gene fusion and modification events. An intriguing question is, whether recent genes encoding such a "halftransporter" still exist as independent folding units. Although it seems likely that the evolution of membrane transport proteins, which harbor internal repeats, is linked to these minimal structural building blocks, their identification in the absence of structural data represents a major challenge, as sequence homology is not an issue. In this review we discuss two protein families, the DedA-family and the SWEET-family, being potential half-transporters and putative ancestors for two of the most abundant secondary transporter families, the MFS family and the LeuT-fold family.
    No preview · Article · Oct 2014 · Biological Chemistry
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    • "Following years of effort, the first three-dimensional structure of LacY was resolved from X-ray diffraction studies using the conformationally restricted mutant (C154G; Cys 154 ! Gly) (Abramson et al., 2003) and, shortly after, the wild-type structure was also completed (Guan et al., 2007). In side view, the monomer of LacY is heart-shaped, and accounts for a diameter of 6 nm displaying a large internal hydrophobic cavity open to the cytoplasmic side. "
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    ABSTRACT: AbstractFörster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a “spectroscopic ruler”. In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H+ symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L α) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.
    Full-text · Article · Jun 2014 · Molecular Membrane Biology
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